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Characterization of functional protein complexes from Alzheimer's disease and healthy brain by mass spectrometry-based proteome analysis.

ABSTRACT: Alzheimer's disease (AD) is a complex neurodegenerative disorder with impaired protein activities. Proteins in the form of complexes have a ubiquitous role in diverse range of cellular functions. The key challenge is to identify novel disease associated protein complexes and their potential role in the progression of AD pathology. Protein complexes were obtained from AD brain prefrontal cortex and age matched controls by Blue Native-Polyacrylamide Gel Electrophoresis. A proteomic analysis was performed using second dimension SDS-PAGE followed by nano LC-MS/MS. Differentially expressed proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). A total of 13 protein complexes with their interacting proteins were resolved on SDS-PAGE. We identified 34 protein spots and found significant abundance difference between the two experimental samples. IPA analysis revealed degeneration of neurons and cell death as a major consequence of protein dysregulation. Furthermore, focused network analysis suggested an integrated regulation of the identified proteins through APP and MAPT dependent mechanisms. The interacting differentially expressed proteins in AD were found to be part of concomitant signaling cascades terminating in neuronal cell death. The identified protein networks and pathways warrant further research to study their actual contribution to AD pathology.

SUBMITTER: Hasan B

PROVIDER: S-EPMC8260596 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionAlzheimer's disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline. Protein biomarkers of AD brain pathology, including ?-amyloid and Tau, are reflected in cerebrospinal fluid (CSF), yet the identification of additional biomarkers linked to other brain pathophysiologies remains elusive. We recently reported a multiplex tandem-mass tag (TMT) CSF proteomic analysis of nearly 3000 proteins, following depletion of highly abundant proteins and off-line fractionation, across control and AD cases. Of these, over 500 proteins were significantly increased or decreased in AD, including markers reflecting diverse biological functions in brain. Here, we use a targeted mass spectrometry (MS) approach, termed parallel reaction monitoring (PRM), to quantify select CSF biomarkers without pre-depletion or fractionation to assess the reproducibility of our findings and the specificity of changes for AD versus other causes of cognitive impairment.MethodWe nominated 41 proteins (94 peptides) from the TMT CSF discovery dataset, representing a variety of brain cell-types and biological functions, for label-free PRM analysis in a replication cohort of 88 individuals that included 20 normal controls, 37 clinically diagnosed AD cases and 31 cases with non-AD cognitive impairment. To control for technical variables, isotopically labeled synthetic heavy peptide standards were added into each of the 88 CSF tryptic digests. Furthermore, a peptide pool, representing an equivalent amount of peptide from all samples, was analyzed (n?=?10) across each batch. Together, this approach enabled us to assess both the intra- and inter-sample differences in peptide signal response and retention time.ResultsDespite differences in sample preparation, quantitative MS approaches and patient samples, 25 proteins, including Tau, had a consistent and significant change in AD in both the discovery and replication cohorts. Validated CSF markers with low coefficient of variation included the protein products for neuronal/synaptic (GDA, GAP43, SYN1, BASP1, YWHAB, YWHAZ, UCHL1, STMN1 and MAP1B), glial/inflammation (SMOC1, ITGAM, CHI3L1, SPP1, and CHIT1) and metabolic (PKM, ALDOA and FABP3) related genes. Logistical regression analyses revealed several proteins with high sensitivity and specificity for classifying AD cases from controls and other non-AD dementias. SMOC1, YWHAZ, ALDOA and MAP1B emerged as biomarker candidates that could best discriminate between individuals with AD and non-AD cognitive impairment as well as Tau/?-amyloid ratio. Notably, SMOC1 levels in postmortem brain are highly correlated with AD pathology even in the preclinical stage of disease, indicating that CSF SMOC1 levels reflect underlying brain pathology specific for AD.ConclusionCollectively these findings highlight the utility of targeted MS approaches to quantify biomarkers associated with AD that could be used for monitoring disease progression, stratifying patients for clinical trials and measuring therapeutic response.

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Characterization of functional protein complexes from Alzheimer's disease and healthy brain by mass spectrometry-based proteome analysis.

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